Biology Reference
In-Depth Information
flavin mononucleotide (FMNH
2
) and myristyl aldehyde to myristic acid and FMN,
a reaction that liberates light at 490 nm (
Waidmann
et al.
,2011
). The
luxAB
genes
encode the heterodimeric luciferase while the
luxCDE
genes encode the enzymes
required for generation of the myristyl aldehyde substrate from myristol ACP
(Waidmann
et al.
, 2011). An important consideration when choosing the type of lucif-
erase to use is that the firefly enzyme requires exogenous addition of the decanal sub-
strate, whereas the bacterial system can be engineered (by inclusion of
luxCDE
in the
operon) to generate the substrate endogenously. The suitability of luciferase systems
for determining promoter activity in real time resides in the instability of the enzyme.
Firefly luciferase is unstable in
B. subtilis
with a half-life of only
6 min (
Mirouze
et al.
,2011a,b
). Thus the suitability of GFPmut3 (half-life of
10 h) and firefly lucif-
erase (half-life or
6 min) for high-resolution kinetic analysis of promoter activity
emanates from their contrasting stabilities
in vivo
. A particular advantage of luciferases
is that most cells are not luminescent so that high signal-to-noise ratios can be
achieved. This makes them particularly suited to the analysis of promoters with low
activity. The necessity for molecular oxygen to generate luminescence is a disadvan-
tage of luciferase reporters, essentially precluding their use as reporters in obligate
anaerobic bacteria. The usage of energy (ATP) and reducing equivalents (FMNH
2
)
by firefly and bacterial luciferases, respectively, might also complicate the measure-
ment or interpretation of promoter activity in metabolic studies.
Despite these limitations, both GFP and luciferases are versatile reporter proteins
that are widely used in expression studies to determine promoter activity. Both
allowglobal promoter activity tobeestablishedwithhigh temporal resolutionand repro-
ducibility without the need to sample or perturb the system under study in any way.
4
CELLULAR PLACEMENT OF TRANSCRIPTIONAL FUSIONS
Placement of the fusion within the cell is an important consideration when planning a
global study of promoter activity. Fusions can be placed on replicating plasmids or
integrated into the chromosome at homologous or heterologous locations. Placing
the fusion on a plasmid makes it easy to mobilize between strains but has the potential
disadvantages of plasmid loss through structural or segregational instability and of var-
iation in copy number, all of which will influence the level of promoter activity. Alter-
natively, fusions can be integrated into the chromosome by a Campbell-type single
crossover event at the locus homologous to the promoter region under study.
A DNA fragment containing the entire promoter region with its 3
0
-end located
upstream of the ribosome binding site of the first gene of the operon under study is
usually chosen as the promoter-containing fragment (
Botella
et al.,
2010, 2011
). This
type of integration is particularly suited to high-throughput generation and analysis of
promoter fusions where plasmid construction and transformation can be automated.
A limitation of this approach is that Campbell-type integrations can result in the pro-
moter fusion being present in multiple copies on the chromosome. This can be caused
by amplification of the integrated plasmid through unequal crossing-over at the
